Vertebrate tinman homologues XNkx2-3 and XNkx2-5 are required for heart formation in a functionally redundant manner

Abstract

Tinman is a Drosophila homeodomain protein that is required for formation of both visceral and cardiac mesoderm, including formation of the dorsal vessel, a heart-like organ. Although several vertebrate tinman homologues have been characterized, their requirement in earliest stages of heart formation has been an open question, perhaps complicated by potential functional redundancy of tinman homologues. We have utilized a novel approach to investigate functional redundancy within a gene family, by coinjecting DNA encoding dominantly acting repressor derivatives specific for each family member into developing Xenopus embryos. Our results provide the first evidence that vertebrate tinman homologues are required for earliest stages of heart formation, and that they are required in a functionally redundant manner. Coinjection of dominant repressor constructs for both XNkx2-3 and XNkx2-5 is synergistic, resulting in a much higher frequency of mutant phenotypes than that obtained with injection of either dominant repressor construct alone. Rescue of mutant phenotypes can be effected by coinjection of either wild-type tinman homologue. The most extreme mutant phenotype is a complete absence of expression of XNkx2-5 in cardiogenic mesoderm, an absence of markers of differentiated myocardium, and absence of morphologically distinguishable heart on the EnNkxHD-injected side of the embryo. This phenotype represents the most severe cardiac phenotype of any vertebrate mutant yet described, and underscores the importance of the tinman family for heart development. These results provide the first in vivo evidence that XNkx2-3 and XNkx2-5 are required as transcriptional activators for the earliest stages of heart formation. Furthermore, our results suggest an intriguing mechanism by which functional redundancy operates within a gene family during development. Our experiments have been performed utilizing a recently developed transgenic strategy, and attest to the efficacy of this strategy for enabling transgene expression in limited cell populations within the developing Xenopus embryo.